1. Field of the Invention
This invention is in the field of biochemistry and more specifically relates to an enzymatic noninvasive method for detecting cancer in mammalian tissue.
2. Description of the Prior Art
New methods for the diagonsis or prognosis of cancer, particularly in human beings, are clearly needed. In bladder cancer, for example, the most widely used method is histopathological and employs a cystoscope. Crystoscopy is costly, involves hospitalization of the patient, and is invasive which presents a certain risk of morbidity. A marker or test for bladder cancer which is not invasive is clearly desirable, particularly for low grade tumors which typically show no readily discernible symptoms.
Normal human bladder epithelium, sometimes called transitional epithelium, is 3-4 cell layers thick and consists of basal cells which are small and cubodial in shape, larger intermediate cells, and very large, highly differentiated cells at the outer, luminal surface. Hicks, The Mammalian Urinary Bladder, Biol. Rev. Cambridge Phil. Soc. 50, 215 (1975). The luminal membrane of these outer cells is unique: it consists of rigid plaques with a highly ordered substructure, separated by unstructured hinge regions. Chemically, this membrane has been found to consist mainly of lipid, with galactoside as one of the polar lipids and glycoprotein as the intrinsic protein embedded in the membrane. Histochemically, this membrane stained for the sugar components of the glycoprotein (Schiff's reagent) and for the negative charges of, presumably, sialic acid (Alcian Blue), the usual end-sugar of negatively charged glycoproteins.
When bladder cancer is induced in experimental animals, extensive changes occur in this surface membrane. The most productive animal model used is the Fischer rat, treated with 0.2% FANFT in the diet to give a high yield of exclusively bladder tumors. Cohen, Jacobs, Arai, Johansson, and Friedell; Early Lesions in Experimental Bladder Cancer: Experimental Design and Light Microscopic Findings, Cancer Research 36: 2508-2511 (1976).
It has been shown that after a 2-6 week treatment with the carcinogen, the normal, luminal surface is retained. Jacobs, Arai, Cohen and Friedell; Early Lesions in Experimental Bladder Cancer: Scanning Electron Microscopy of Cell Surface Markers, Cancer Research 36: 2512-2517 (1976). If at this time the carcinogen is withdrawn, no tumors ensue, although exfoliative cells are released as a result of hyperplasia, revealing the underlying surface of the intermediate cells, covered with uniform microvilli. After 8 weeks, microvilli of varying thickness and length appear. If the carcinogen is then withdrawn, hyperplasia continues with pleomorphic microvilli appearing on the luminal surface even after 50 weeks. If the carcinogen is fed 10 weeks or more, invasive carcinomas are formed, covered by pleomorphic microvilli. There is, therefore, a time-point of irreversibility in the carcinogenic process, coinciding with the appearance of the pleomorphic microvilli. Similar observations were made by others, in addition to the discovery of glycocalyx which covered the microvilli. Hicks and Wakefield; Membrane Changes During Urothelial Hyperplasia and Neoplasia, Cancer Research 36: 2502-2507 (1976).
A glycocalyx covering the microvilli of a luminal surface is also found in the intestinal and kidney mucosa. It is considered to be a network of enzymes covering the plasma membrane of the microvilli and consists of glycoprotein material, being labeled by sugar precursors (glucose, glucosamine, galactose, mannose). Ito, Structure and Function of the Glycocalyx; Fed. Proc. 28: 12 (1969).
It is clear, then, if only considered from a morphological and histochemical viewpoint, malignant transformation is accompanied by changes of the plasma membrane in the bladder epithelium involved. Glycoproteins are part of these membranes. Changes in specific glycoproteins of plasma members as a result of transformation have been well documented biochemically. Hynes, Cell Surface Proteins and Malignant Transformation. Biochim. Biophy. Acta 458: 73-107 (1976).
At the same time, recent evidence implicates the enzymes which are necessary in the synthesis of glycoproteins. In fact, Roseman proposed the involvement of cell-surface glycosyl transferases in contact inhibition of normal cell growth, and, of course, loss of such inhibition in transformed cells. Roseman, Chem. Phys. Lipids, 5: 270-297 (1970). Cell-surface galactosyl transferase has been detected in lymphocytes and other cells. Cacan, Verbert and Montreuil, New Evidence for Cell Surface Galactosyltransferase. FEBS Letters 63: 102-106 (1976). Porter and Bernacki, Nature, 256: 748-650 (1975). Recently, Weiser et al. found serum gal transferase in 58 patients with various types of carcinoma to increase almost 2-fold, and showed the presence of a gal transferase isoenzyme exclusively in the cancer patients. Weiser, Podolsky and Isselbacher, Cancer-associated Isoenzyme of Serum Galactosyltransferase. Proc. Nat. Acad. Sci. 73: 1319-1322 (1976). A 3-5 fold increase in this enzyme was detected in the serum of ovarian cancer patients. Galactosyl Transferase in Ovarian Cancer Patients, Cancer Research 36: 2096 (1976). Though the mechanism of the involvement of glycoproteins and glycosyl transferases in the malignant transformation is not understood, it is clear that there is an intimate connection between those compounds and enzymes, and the process of carcinogenesis.